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Pharmacological inhibition of PSPH reduces serine levels and epileptic seizures

Nature Chemical Biology (2025)Cite this article

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Abstract

Temporal lobe epilepsy (TLE) is the most common type of drug-resistant epilepsy. Lowering the levels of N-methyl-d-aspartate receptor (NMDAR) ligands has been suggested as a promising therapeutic strategy for TLE. d-Serine gates synaptic NMDARs in the hippocampus but the effect of d-serine on seizure activity remains poorly understood. Here, we show that serine levels in the hippocampus were increased in persons with TLE and in a mouse model of TLE. Eliminating d-serine or blocking its binding with NMDARs suppressed seizures in mouse models. Astrocyte-derived l-serine was found to regulate interstitial d-serine levels and seizure activity through a process controlled by phosphoserine phosphatase (PSPH). We identified a potent PSPH inhibitor, Z218484536, and found that its systemic administration reduced spontaneous epileptic discharges in mouse and cynomolgus monkey models of TLE. Overall, these results indicate that PSPH is a promising therapeutic target for TLE and support further preclinical studies of Z218484536.

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Fig. 1: d-Serine promotes KA-induced acute seizures.
Fig. 2: Depletion of hippocampal D-serine inhibited 4-AP-induced PDS in CA2 neurons.
Fig. 3: Astrocyte-derived l-serine regulates d-serine levels and seizure activity.
Fig. 4: PSPH is upregulated in TLE and regulates extracellular l-serine and d-serine and seizures.
Fig. 5: Virtual screening and experimental validation identified new PSPH inhibitors.
Fig. 6: Z218484536 has antiseizure effects in the KA mouse model of chronic TLE.

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Data availability

The structure of Z218484536 is accessible from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) under compound identifier 136130484. The proteomic and phosphoproteomics raw data were deposited to the ProteomeXchange Consortium through the iProX repository with the dataset identifiers PXD061834 and PXD061835. The original metabolomics data were deposited to MetaboLights under identifier MTBLS12322. Source data are provided with this paper.

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Acknowledgements

This work was supported by research grants from the National Natural Science Foundation of China (81930104 to Q.X., 82171447 to L.S. and 32293213 to Q.X.), Ministry of Science and Technology of the People’s Republic of China (2021ZD0203001 to Q.X., 2022ZD0211700 to Q.X., 2021ZD0200600 to Q.X. and 2021ZD0202000 to Q.X.), National Key Research and Development Program of China (2020YFA0804502 to Q.X.), CAMS Innovation Fund for Medical Sciences (2021-I2M-1-020 to Q.X.) and Fundamental Research Funds for the Central Universities, PUMC (3332024218 to Q.X.).

Author information

Author notes

  1. These authors contributed equally: Longze Sha, Yanbing Wang.

Authors and Affiliations

  1. State Key Laboratory of Common Mechanism Research for Major Diseases, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Longze Sha, Yanbing Wang, Peixin Meng, Yu Deng, Xiuneng Zhang & Qi Xu

  2. State Key Laboratory of Complex, Severe and Rare Diseases, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China

    Longze Sha

  3. Neuroscience Center, Chinese Academy of Medical Sciences, Beijing, China

    Longze Sha, Yousong Ye & Qi Xu

  4. Institute of Child and Adolescent Health, School of Public Health, Peking University, Beijing, China

    Ting Chen

  5. Institute of Medical Biology, Chinese Academy of Medical Sciences, Kunming, China

    Yousong Ye

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Contributions

L.S. and Y.W. performed most of the molecular biological, electrophysiological and animal experiments and analyzed the data. P.M. and X.Z. performed the antiseizure experiments in mouse models. Y.D. conducted the immunohistochemical assay. L.S., Y.W., T.C. and Y.Y. performed the antiseizure experiments in the monkey model. X.Z. and T.C. participated in the behavioral data analysis. L.S., Y.W. and Q.X. designed the research and wrote the manuscript.

Corresponding author

Correspondence to Qi Xu.

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Competing interests

Q.X., L.S. and Y. W. are inventors on a patent pending for the use of PSPH inhibitors to treat epilepsy (202411346150.8/WP240041BJ). The other authors declare no competing interests.

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Nature Chemical Biology thanks David Sabatini and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 D-serine regulates spontaneous recurrent seizures.

(a) Schematic diagram depicting the protocols for modifying D-serine levels in the hippocampus of mice 5 weeks after the establishment of KA-induced SE (n = 5 for the glycine group and n = 8 for the other groups). (b-p) Representative 1-min EEG tracings (left), schematic illustrations of seizure event distribution (middle; each seizure event (HPD) is shown as a tick mark), and bar plots showing seizure event frequency at baseline and 1 h and 12 h after unilateral hippocampal injection of vehicle (b-d), DAAO (e-g), 7-CKA (h-j), D-serine (k-m), or glycine (n-p) (right). After a 20-h baseline EEG recording, the mice were injected with 0.5 μL of vehicle, DAAO (1 U/ml), 7-CKA (100 μM), D-serine (10 μM), or glycine (200 μM) into the hippocampus ipsilateral to the KA injection via a preimplanted cannula at 8:00 on day 2. Statistical comparisons of seizure frequency (number of HPDs per hour) were performed via two-tailed paired t tests. Baseline: from 12:00 on day 1 to 8:00 on day 2; 0–12 h postinjection, 8:00 to 20:00 on day 2. 12–28 h postinjection, 20:00 on day 2 to 12:00 on day 3. Inj., injection. The data are shown as the means with individual points.

Source data

Extended Data Fig. 2 7-CKA and DAAO have limited effects on 4-AP-induced PDS in DG and subiculum neurons.

Electrophysiological data recorded from DG and subiculum neurons in hippocampal slices of normal. (a-h) and TLE mice (n = 8) (i-p). The data are shown as the means with individual points. Statistical comparisons of the area and amplitude of PDSs following exposure to 4-AP and 4-AP + 7-CKA (100 μM) and washout of 7-CKA (retaining 4-AP) were performed with the two-tailed paired t test (a, b, e, f, i, j, m, n). Statistical comparisons of the area and amplitude of 4-AP-induced PDS in control slices and DAAO-treated (1 U/ml) slices were performed with two-tailed Student’s t test (c, d, g, h, k, l, o, p).

Source data

Extended Data Fig. 3 Effects of exogenous D-serine on 4-AP-induced PDS.

(a) Representative traces of whole-cell patch recordings from mice receiving injections of 10 μM D-serine. (b, c) Statistical comparisons of the area and amplitude of PDS following exposure to 4-AP and 4-AP + low-dose (LD) D-serine (10 μM) and after washout of D-serine (retaining 4-AP) (n = 5). (d) Representative traces of whole-cell patch recordings from mice treated with 100 μM D-serine. (e, f) Statistical comparisons of the area and amplitude of PDS following exposure to 4-AP and 4-AP + high-dose (HD) D-serine (100 μM), and after washout of D-serine (retaining 4-AP) (n = 6). The data are shown as the means with individual points. Statistical analyses were performed with two-tailed paired t tests.

Source data

Extended Data Fig. 4 The levels of PHGDH and PSAT are unchanged in TLE.

(a-d) Screening of siRNAs against PHGDH, PSAT1 and PSPH. Immunoblot (a) and statistical analysis (b-d) of cultures of astrocytes transfected with the indicated siRNA. (e, f) Statistical analysis of L-serine levels in the cell supernatant (e) and lysates (f) of cultures of astrocytes following PSPH, PSAT1 or PHGDH KD with another set of siRNAs (n = 4). (g, h) Immunofluorescence staining for PHGDH (g) or PSAT1 (h) in hippocampus samples surgically resected from TLE patients with hippocampal sclerosis (TLE-HS) and autopsy controls without neurological disorders (control). (i, j) Immunofluorescence staining of PHGDH (i) or PSAT1 (j) in hippocampus samples from control and TLE mice (4 weeks post-KA-induced SE); scale bar = 50 μm. Statistical analyses were performed with one-way ANOVA followed by Tukey’s post hoc comparison test (e, f) or two-tailed Student’s t test (g-j).

Source data

Extended Data Fig. 5 Molecular docking, PK and toxicological data for Z218484536.

(a) Docking score for the top 20 hits for PSPH inhibitors. (b) Binding mode and molecular interaction between Z218484536 and PSPH. (c) MST analysis of the binding of Z218484536 to PSPH. (d) In vivo PK data in mice after IP injection of a single dose of 4 mg/kg Z218484536. (e) PK profile of Z218484536 in the plasma and brain following a single IP administration of 4 mg/kg Z218484536 (n = 3 per time point). (f) Calculation of the brain/plasma ratio for Z218484536. (g, h) CCK-8 assays for Z218484536 (g) and LTG (h) in HepG2 cells. The cells were treated with each compound for 48 h before the CCK-8 assays were performed. (i) LD50 toxicity test for Z218484536. LD50 data are the averages from three independent experiments (n = 10 mice per group). (j-m) Performance of mice administered Z218484536 (4 mg/kg) or vehicle by IP injection for seven consecutive days in the open field test (j, k) and rotarod test (l, m) (n = 8 per group). (n, o) L-serine and glycine levels in the serum of mice administered Z218484536 (4 mg/kg, n = 6) or vehicle (n = 4) by IP injection for seven consecutive days. The data are shown as the means ± SDs or means with individual points. Statistical analysis was performed via two-tailed Student’s t test.

Source data

Extended Data Fig. 6 Target engagement of Z218484536.

(a) PSPH protein sequence alignment between 11 species showing high evolutionary conservation of the four PSPH binding sites (arrowheads). (b) Assays of PSPH activity for wild-type (WT) and carriers of the Asp22Ala, Ala51Val, Ala51Gly, Gly110Val, Gly110Ala, Lys158Ala or Ala51Val/Gly110Ala mutations. (c) MST analysis of Z218484536 binding to the PSPH Ala51Val/Gly110Ala mutant. (d) Immunoblot analysis of cultured astrocytes after lentivirus-mediated PSPH-MT (Ala51Val/Gly110Ala) OE or PSPH KD or their combination with or without Z218484536 treatment. The sequence encoding PSPH-MT was optimized to avoid interference by shRNAs that target the endogenous PSPH gene sequence. (e, f) Levels of L-serine in cellular lysates (e) and supernatants (f). (g, h) The effects of Z218484536 in mice after bilateral hippocampal injection of AAV5-gfa104-miPSPH-eGFP to silence PSPH gene expression. One month after AVV injection, the mice were injected with Z218484536 once a day for three consecutive days (4 mg/kg), and PTZ (55 mg/kg) was used to induce acute seizures. (i, j) Statistical comparisons of time to first seizure (onset; i) and the total number of seizures per mouse (j) among the four groups. The mice were injected with vehicle or 4 mg/kg Z218484536 daily for three days. Awake mice were preinjected with 0.25 μL of vehicle or D-serine (10 μM) into the unilateral hippocampus though a preimplanted guide cannula, followed by injection of 7 ng of KA (0.25 μL). The data are shown as the means ± SDs or means with individual points. Statistical analyses were performed with one-way ANOVA followed by Tukey’s multiple comparisons post hoc test (b, e, f), two-tailed Student’s t test (g, h) and two-way ANOVA followed by Tukey’s multiple comparisons post hoc test (i, j).

Source data

Extended Data Fig. 7 Z218484536 inhibits epileptic spikes in a cynomolgus monkey model of TLE.

(a) Experimental design. The control group consisted of Control-1 (female, 3.7 kg, 7 years old), Control-2 (male, 6.5 kg, 6 years old), and Control-3 (male, 6.9 kg, 7 years old); the Z218484536 group consisted of Z218484536-1 (female, 3.6 kg, 7 years old), Z218484536-2 (6.1 kg, 10 years old), and Z218484536-3 (male, 7.7 kg, 9 years old). (b) Representative 1-min EEG tracings showing EEG activity before and after administration of vehicle or Z218484536. (c) Spike frequencies at baseline (yellow background, 3 EEG recordings) and during treatment with vehicle and Z218484536 (green background, 4 EEG recordings). (d, e) Statistical comparisons of spike frequencies among baseline and time points 1 and 4 after treatment with vehicle (d) or Z218484536 (e). (f) The plasma concentration of Z218484536 in monkeys 1 h after treatment. Data were obtained for the three Z218484536-treated monkeys. The data are shown as the means with individual points. Statistical analyses were performed via two-tailed paired t test (d, e).

Source data

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Supplementary Figs. 1–6, Notes 1 and 2 and Table 5.

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Supplementary Tables 1–4

Supplementary Tables 1–4 as referenced in the main manuscript.

Supplementary Data 1

Summary of all supplementary raw data.

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Sha, L., Wang, Y., Meng, P. et al. Pharmacological inhibition of PSPH reduces serine levels and epileptic seizures. Nat Chem Biol (2025). https://doi.org/10.1038/s41589-025-01920-5

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